Increased homogeneity of mycological biopolymer grown into void space

ABSTRACT

The method of growing a biopolymer material employs incubation of a growth media comprised of nutritive substrate and a fungus in containers that are placed in a closed incubation chamber with air flows passed over each container while the chamber is maintained with a predetermined environment of humidity, temperature, carbon dioxide and oxygen. The air flows may be directed parallel or perpendicularly to the surfaces of the growth media.

This is a Non-Provisional Pat. Application and is a continuation of U.S. Application No. 16/190,585, filed Nov. 14, 2018, which claims the benefit of Provisional Pat. Application 62/707,704, filed Nov. 14, 2017. Any and all applications for which a foreign or domestic priority claims is identified in the Application Data Sheet as filed with the present application are hereby incorporated by reference under 37 C.F.C. § 1.57.

This invention relates to methods to create a biomaterial of increased homogeneity, strength and density as compared to the mycological biopolymer described in published U.S. Pat. Application US 2015/0033620 (A).

As described in published U.S. Pat. Application US 2015/0033620 (A), the environmental conditions for producing the mycological biopolymer product, i.e. a high carbon dioxide (CO₂) content (from 5% to 7% by volume) and an elevated temperature (from 85° F. to 95° F.), prevent full differentiation of the fungus into a mushroom. There are no stipe, cap, or spores produced. The elevated temperature accelerates tissue production. The biopolymer product grows into the void space of the tool, filling the space with an undifferentiated mycelium chitin-polymer, which is subsequently extracted from the substrate and dried.

Briefly, the invention allows for the production of a tough, pliable material that could be used to replace leather, leather-like materials, textiles and high density and strength foams in many applications such as upholstery, apparel/fashion, military gear, athletic gear, and footwear.

The invention involves growing a mycological biopolymer under conditions of directed airflow, depositing moisture and solutes, such as minerals, on the surface of the growing organism, growth through a scrim or lofted non-substrate matrix, and fluctuation of the humidity profile throughout growth to induce more homogenous material and produce a range of material densities. The mycological biopolymer product consists entirely of fungal mycelium.

One embodiment of the invention is the placement of contained inoculated growth media used to produce mycological biopolymer within a growth enclosure equipped to deliver a directed airflow across at least one of the surfaces of the growth media.

In this embodiment, the method of growing a biopolymer material comprises the steps of providing a plurality of containers, each of which defines a cavity containing a growth media comprised of nutritive substrate and a fungus; placing the containers in a closed incubation chamber; maintaining the incubation chamber with a predetermined environment of humidity, temperature, carbon dioxide and oxygen sufficient to produce a mycelium biopolymer while preventing full differentiation of said fungus into a mushroom; directing flows of air containing a high carbon dioxide content through the incubation chamber for passage over the growth media in each container; and incubating the growth media in each container for a period of time sufficient for the fungus to digest the nutritive substrate and produce a mycelium biopolymer consisting entirely of fungal mycelium in each container.

Each container may be placed within the incubation chamber within an “airflow box” such that the height of the container interacts with the airflow or each container may be sunk into the airflow box such that the total cross-sectional area of the box can be employed.

In accordance with the invention, the flows of air are directed into the closed incubation chamber laterally of the containers or perpendicularly of the containers.

A second embodiment of the invention employs the controlled deposition of moisture and minerals on at least one of the growing surfaces to induce homogeneity with a range of densities based on the moisture and mineral deposition volume.

In this embodiment, the method of growing a biopolymer material comprises the steps of providing a plurality of containers, each of which defines a cavity containing a growth media comprised of nutritive substrate and a fungus; placing the plurality of containers in a closed incubation chamber; maintaining the incubation chamber with a predetermined environment of humidity, temperature, carbon dioxide and oxygen sufficient to produce a mycelium biopolymer while preventing full differentiation of said fungus into a mushroom; distributing a mist through the incubation chamber for passage over the growth media in each container; and incubating the growth media in each container for a period of time sufficient to produce a mycelium biopolymer in each container.

In accordance with the invention, the mist includes moisture and a solute, such as minerals.

A third embodiment of the invention involves the growth of a mycological biopolymer through a scrim or lofted non-substrate matrix that is in direct contact or elevated above the substrate growth surface and grown in a container without the use of a lid.

A fourth embodiment employs the fluctuation of the percent humidity at time periods of growth throughout the duration of the cycle in order to induce a higher density material of increased homogeneity.

A fifth embodiment uses specific air flow rates to achieve a range of aerial mycelium densities and mechanical performances.

In all the embodiments of the invention, the mycological biopolymer is grown from a nutritious substrate, and grows into a panel at a dry density of 0.5 to 4 pounds per cubic foot. The localized environmental conditions, i.e. high carbon dioxide air, moisture deposition and temperature, must be homogenous, except for the embodiment using a scrim or lofted non-substrate matrix, in order to achieve uniform growth within each panel and throughout the larger growing chamber.

As further described in published U.S. Pat. Application US 2015/0033620 (A) the use of a lid was enlisted to control the localized environmental conditions influencing the growth of the mycological biopolymer.

In accordance with the invention, under directed airflow, the lid on the container is removed and the localized environmental conditions are homogenized via airflow. The use of airflow allows for growth from the full surface of the growth container and helps to improve the homogeneity and uniformity of the tissue grown. This may be attributed to the airflow facilitating the delivery of humidity, water and solutes, such as minerals, to the growing tissue, elimination of microenvironments, and/or increased mechanical force. There are many applications for a biological textile and foam that require increased volume of homogenous material.

The growth environments used in the production of edible mushrooms, both specialty and Agaricus currently employ the use of some uncontrolled airflow through the growth chambers for heating, cooling, of gassing carbon dioxide produced by the growing mushrooms or introducing oxygen into the growing chamber. This differs from the airflow technology employed to prevent any and all differentiation of the fungus into a fruiting body that makes an edible mushroom while providing a uniform environment to grow mycological biopolymer

Further, airflow within the cultivation of mushrooms is directed at removing metabolic byproducts such as carbon dioxide and other volatiles, and is intermittent in nature. The airflow employed to grow mycological biopolymer is directed at providing a consistent homogenization of the incubation environment without localized variations that has sufficiently controlled parameters (e.g., high carbon dioxide) such that the mycelium cannot differentiate into a mushroom. Also, the airflow velocity provides a directed force that modulates the structure of the aerial mycelium, impacting density.

While the growth environments used in the production of edible mushrooms can employ the use of an airflow through the growth chambers, the air flow is indirect and part of a recirculating system for humidification of the environment. The airflow is not directed across the surface of the growth media as is the case in accordance with the invention.

These and other objects and advantages will become more apparent from the following detailed description taken with the accompanying drawings wherein:

FIG. 1A illustrates photographs of the top surfaces of panels grown in a direct, high airflow environment with minimal differentiation in tissue morphology in accordance with the invention;

FIG. 1B illustrates photographs of the top surfaces of panels grown in an indirect, low airflow environment with highly differentiated tissue;

FIG. 1C illustrates photographs of the top surfaces of panels grown in a zero-airflow environment and resulting in highly differentiated tissue and reduced aerial growth;

FIG. 2 illustrates a chart of treatment versus density in accordance with the invention;

FIG. 3A1 schematically illustrates a lateral airflow system in accordance with the invention;

FIG. 3A2 illustrates a perspective view of an air box used for the incubation of two containers in accordance with the invention

FIG. 3B schematically illustrates a modified lateral airflow system in accordance with the invention;

FIG. 3C schematically illustrates another modified lateral airflow system in accordance with the invention;

FIG. 4A schematically illustrates a perpendicular airflow system for passing air over the surface of the growth medium in accordance with the invention;

FIG. 4B illustrates a photograph of the top surface of a panel grown in the system of FIG. 4A;

FIG. 4C schematically illustrates the air flow patterns over a growth medium in the system of FIG. 4A;

FIG. 5A schematically illustrates a mist distribution system in accordance with the invention; and

FIG. 5B schematically illustrates an indirect air flow system for recirculation of humidified air not in accordance with the invention.

Referring to FIG. 3A1 , in a first embodiment, the method of growing a biopolymer material employs a closed incubation chamber 10 having a plurality of vertically spaced apart shelves 11 and transparent front walls (not shown) for viewing the interior of the chamber 10.

In addition, an air flow system 12 is connected with the chamber 10 for directing air flows laterally across the chamber 10 as indicated by the arrows 13 from one side of the chamber 10 to and through the opposite side of the chamber 10. As illustrated, the air flow system 12 includes a manifold M in the upper part of the chamber 10 for distributing humidified air across the top of the chamber 10 for cascading down the shelves 11 until being recirculated on the bottom right for re-humidification.

Each shelf 11 of the chamber 10 is sized to receive an air box B that contains two containers 14 each of which contains a growth media 15 comprised of nutritive substrate and a fungus.

Referring to FIG. 3A2 , each container 14 is in the form of a rectangular tray with an open top to define a cavity of a size of 11.5 inches by 18.5 inches with a 1 inch lip around the entire container that extends externally outwardly of the cavity. Each container is placed within the air box B.

The containers 14 are constructed from a sufficiently rigid, non-reactive material, such as polycarbonate, and the orifice of the container is such that it is paired with the airflow device to achieve the desired air flow rates. The length of the container along with the airflow rates dictate the consistency of this flow, and the entrance length before the airflow reaches the growing part is impart to control the laminar or turbid nature of the flow. The containers can include ramps, fairings, such as airfoils, or baffles, to assist in homogenizing the flow.

The air box B is of rectangular shape that receives the growth trays 14 and has an open side 16 in one end face and a smaller orifice 17 in an opposite end face.

The air flow system 12 includes a fan 12' situated at the orifice 17 of each air box B to pull air over the growth media 15 in the containers 14 and growing part as indicated by the horizontal arrows. The orifice is covered by the fan to ensure all of the air moves through the fan. Alternatively, the fan 12' may be positioned at the open side 16 of the air box B to push air over the growth media 15.

As indicated, the humidified air cascading down from the manifold M passes into and through each air box B via the orifices 16, 17.

Specifically, the growth media 15 comprises: Materials Input Approximate Materials Amount

-   Bagged Sealed Substrate:     -   Corn Stover 6000 g     -   Poppy Seeds 1440 g     -   Maltodextrin 256 g     -   Calcium sulfate 80 g     -   Municipal water 16000 g

-   Inoculant:     -   Ecovative Strain ID 2880 g     -   045-08-003 spawn

During the method of growing a biopolymer material, the incubation chamber 10 is maintained with a predetermined environment of humidity, temperature, carbon dioxide and oxygen. Specifically, the chamber 10 is maintained at 99% relative humidity (RH), 5% CO₂, and a fluctuating temperature of from 85° F. to 90° F. during the step of incubating.

The incubation chamber 10, i.e. growth enclosure, can be open on one end and on the other can be outfitted with fans or apparatuses for moving air over the containers 14 in a lateral direction as indicated by the arrows 13 either by pulling or pushing air at speeds ranging from 5 CFM to 10,000 CFM steadily or in a pulsing fashion. The incubation chamber 10 can be within a larger incubation chamber (not shown) that is able to maintain environmental conditions including humidity, temperature, carbon dioxide and oxygen.

The shape and construction of the incubation chamber 10 can be specially crafted to assist in directing the air flow and laminar or turbid characteristics of the air flow.

PROCESS STEPS (SEE FIG. 3A1) Directed Lateral Airflow

-   1. Nutritious growth media and organism inoculum 15 is packed into     containers 14 as described in US 20150033620 A with the exception     that these containers 14 are not outfitted with lids. -   2. These containers 14 are placed within air boxes B on the shelves     11 of the enclosed incubation chamber 10. -   3. Directing flows of air via the airflow system 12 through the     incubation chamber 10 for passage laterally over the growth media 15     in each container 14 as indicated by the arrows 13. -   4. incubating the growth media 15 in each container 14 for a period     of time sufficient to produce a panel P of mycelium biopolymer in     each container 14, e.g. panels can be grown for 4 to 14 days within     the incubation chamber 10.

The flows of air are generated by fans outfitted to the incubation chamber 10 and are directed over the containers 14 and back into the greater incubation space.

Referring to FIG. 1A, a pair of panels 17 produced in accordance with the above method consists entirely of fungal mycelium and show minimal differentiation in tissue morphology.

Airflow rates of 100 cubic feet per minute at a constant RH of >99% resulted in tissue with a dry density of 1.98 pcf and a tensile strength of 17.5 psi. These panels offered a high degree of consistency.

Airflow rates of 100 ― 175 cubic feet per minute and relative humidity drop to 96% for a period of 48 hours resulted in tissue with a dry density of 1.45 pcf and a tensile strength of 13.6 psi. These grown panels resulted in a high degree of consistency.

Airflow speeds of 300 - 350 cubic feet per minute and at a constant RH of >99% resulted in tissue with a dry density of 3.32 pcf and a tensile strength of 31.2 psi.

Referring to FIG. 1B, pairs of panels produced under conditions without a directed airflow were characterized in having highly differentiated tissue.

Referring to FIG. 1C, pairs of panels grown in a zero-airflow environment were characterized in having highly differentiated tissue and reduced aerial growth;

Referring to FIG. 3B, wherein like reference characters indicate like parts as above, the incubation chamber 10 may be constructed with vertically spaced apart shelves 11 (or racks) and may be enclosed by sheeting (not shown) for cooperation with containers 14 of extended length such that each shelf 11 receives an air box B with only a single container 14.

In addition, the incubation chamber 10 is outfitted with a lateral airflow system 12' having fans fitted to the chamber 10' to direct airflow from the incubation environment through the air boxes B and over the containers 14 and back into the greater incubation space as indicated by the arrows 18.

Referring to FIG. 3C, wherein like reference characters indicate like parts as above, the incubation chamber 10' may have open shelves 11 on which containers 14 with growth medium 15 are placed without using air boxes. In addition, the incubation chamber 10' is outfitted with a lateral airflow system having fans (not shown) located on the right-hand side, as viewed, of the chamber 10' for pulling air flows through and out of the chamber 10' while passing laterally over the containers 14.

Referring to FIG. 4A, wherein like reference characters indicate like parts as above, the growth of the mycological biopolymer may be effected by passing the airflows perpendicularly of the containers 14.

For example, the enclosed incubation chamber 10" may be constructed with one or more air flow devices (not shown) positioned above the nutritive media 15 to push or pull conditioned air over the growing mycelium. The air flow device 12 as in FIG. 3A1 is either held static at a desired height above the growth container 14' or modulated on linear actuators (not shown) through the course of growth.

As illustrated, two containers 14' are positioned on each shelf 11 within the incubation chamber 10ʺ and each container 14ʹ is provided with vertical standoffs 18 that space a cover 19 (roof) from a container 14ʹ. The vertical standoffs 18 are fabricated from a non-reactive substance, such as polyvinylchloride (PVC), and are sufficiently rigid to resist the forces of the airflow device.

The incubation chamber 10ʺ, can be open on one end and on the other can be outfitted with fans or apparatuses for moving air over the containers 14ʹ in a direction perpendicular to the growing surface as indicated by the arrows 13ʺ either by pulling or pushing air at speeds ranging from 5 CFM to 10,000 CFM steadily or in a pulsing fashion.

The incubation chamber 10ʺ can be within a larger incubation chamber (not shown) that is able to maintain environmental conditions including humidity, temperature, carbon dioxide and oxygen.

Referring to FIG. 4B, a panel of mycological biopolymer produced in the incubation chamber 10ʺ may be characterized in having a concentration of mycelium below the airflow device as the air was pulled up over the growing surface as indicated in FIG. 4C as opposed to across the growing part in FIG. 1A. As indicated in FIG. 4B, where airflow device pulled the air upwardly from a central region of the growth medium, the growing mycelium was concentrated in the central region of the panel.

Directed Perpendicular Airflow (see Fig. 4A)

-   1. Nutritious growth media and organism inoculum is packed into     containers as described in US 20150033620 A with the exception that     these containers are not outfitted with lids. -   2. These containers 14" are placed within the enclosed incubation     chamber 10". -   3. Directing flows of air via the airflow system 12 through the     incubation chamber 10" for passage perpendicularly of the growth     media in each container 14" as indicated by the arrows 13". -   4. The shape and design of the growth enclosure can be specially     crafted to assist in directing the flow and laminar or turbid     characteristics of the air. -   5. incubating the growth media 15 in each container 14" for a period     of time sufficient to produce a panel of mycelium biopolymer in each     container 14", e.g. panels can be grown for 4 to 14 days within the     incubation chamber 10". -   6. Air movement can be used to mold and structure the material into     particular shapes and patterns during growth for a final product     that is shaped using airflow.

In Step 6 above, pulled horizontal airflow velocity (>175 cfm) creates a dense scalloped pattern. Vertical airflow creates structures below the airflow device presenting a morphology that parities the airflow (pulled upward like a stalagmite). Pushing creates wave patterns opposing the airflow (160 CFM). Proximity to the airflow device and the pattern of airflow generates tissue patterns that mimic the flow.

Referring to FIG. 2 , as graphically illustrated, the moisture and solute content of the growth media has been found to directly relate to the density of the material being grown. The higher the moisture content, the lower the density of the material grown, a trend that has been shown across an assortment of substrate types.

FIG. 2 shows three other substrate varieties in comparison to the corn stover material at 4 different moisture contents. This resulted in variations in the final product density, which higher moisture contents resulting in lower density tissue.

Tukey Kramer is a mean (average) comparison test that determines the significant difference between tests. The 0.05 is the confidence interval, so there is a 95% confidence in the relationship between the data.

The ability for fungal cells to fill the void space is dependent on the water and solutes available to the organism during growth. The more water available, the more aggressively the organism can expand, causing the density of the material to drop.

Accordingly, referring to FIG. 5A, wherein like reference characters indicate like parts as above, an enclosed incubation chamber 20 is fitted with a mist distribution system 21 so that moisture and solutes can be applied to the growing tissue through a number of avenues for the purpose of producing a range of material densities in the produced mycological biopolymer.

As illustrated, the incubation chamber 20 has a plurality of vertically spaced apart shelves 21 and transparent front walls (not shown) for viewing the interior of the chamber 20. The incubation chamber 20 is sized to receive a plurality of containers 14, each filled with a growth media 15.

As above, the incubation chamber 20 can be placed within larger incubation chambers that are able to maintain uniform environmental conditions including humidity, temperature, carbon dioxide and oxygen.

The mist distribution system 21 is positioned to deliver moisture and solutes, such as minerals, to the top of the growing tissue in each container 14 and can also be used to control the material density and regulate the homogeneity of the material. This material is comprised of aerial hypha growing up and out of a nutritious space into a non-nutrient environment. In order to control growth in such an environment, the organism employs the use of turgor pressure to regulate the extension of the hyphae at the apex, or hyphal tip. Thus, regulating the amount, distribution and/or droplet size of available moisture and solutes deposited across the top surface of the growing material can control the osmotic gradient created within the hyphae and subsequently, its growth rate and pattern of colonization.

Solutes are any agent that can cause an osmotic potential. RO (reverse osmosis) or distilled water are free of such agents. Other solutes could include proteins, carbohydrates, polymers, and minerals.

A solute is a material that induces an osmotic potential within a solution. A solute can be a mineral, a carbohydrate, a protein, or lipid. Concentrations of a solute on one side of a membrane, such as a cell membrane and/or wall, will drive a potential across the membrane if the solution on the opposing side of the membrane has a lesser concentration of the solute.

Moisture and solute deposition can be employed to achieve specific material densities and increase material homogeneity.

Moisture and solutes can be distributed across the growing surface of the growth media using a bath of water outfitted with a “humidifying puck” that atomizes the water into vapor or mist. A “humidifying puck” is an ultrasonic humidifier which produces low quality, high liquid content, droplets of a size range of 5 to 22 microns. The liquid water droplet, opposed to vapor, is important as the droplet can carry a solute. The same is true for sprays or bubblers, but cannot be achieved with steam. Steam can be used to regulate humidity, but not as a substitute for water carrying the solutes.

This mist can be distributed across the surface of the growth media using indirect airflow from a fan or similar apparatus or by a spray nozzle that can be outfitted with compressed air or other means of expelling the moisture out of the nozzle and directed at the growing surface of the growth media.

The amount of moisture and minerals, the distribution, and the droplet size can be regulated to produce a homogenous mycelium biopolymer of varying densities.

Fluctuation of the percent humidity during the growth cycle can be employed as a method to increase the density and homogeneity of the material. In the method described in the published US 2015/0033620 A, the humidity was held static throughout the duration of the growth cycle to achieve material growth. By altering this paradigm and fluctuating the humidity of the growth chamber at targeted stages during the growth cycle, the density and homogeneity can be increased.

A moist environment is generally necessary for fungi to grow aggressively. When a desiccating environment is encountered, many species of fungi have developed methods to protect themselves against moisture loss. For aerial hyphae, a localized high humidity environment is necessary to allow for continued expansion and prevent collapse of the hyphae towards the growing surface. Fluctuation of the humidity in the growth chamber can be used to trigger physiological responses of the organism to a desiccating environment as well as to manipulate the aerial hyphal growth in order to achieve the desired material characteristics.

A system design allowing for the controlled deposition of mist onto the growing material without the use of airflow was prototyped and tested employing the incubation chamber of FIG. 5A. This misting system prototype evenly distributed an equivalent volume of mist onto the growing material as a control high airflow system. The misting system used a SF1010SS siphon fed atomizing nozzle, or “atomizer” to expel a fan shaped spray of fine water droplets, equivalent in size to MycoFlex™ control technology as employed in the methods described in US 2015/0033620, across the growing surface of the experimental parts without the use of direct airflow.

The atomizer misting system was set up with the nozzle positioned 26.5 inches in from the incubator wall to the right side of the target growth surface. The nozzle was affixed at a 45-degree angle to the shelf 11 above the target container 14 and rotated 90-degrees, resulting in a vertically oriented fan-shaped spray pattern. The target total volume of moisture of 0.28 microsiemens per centimeter (uS/cm) per minute plus/minus seven microsiemens per centimeter (uS/cm) as well as target deviation in moisture across the panel surface of 0.00014 g/min was achieved using a misting paradigm of 2.4% time misting over a 1 minute period. The target volume was based on TDS values collected for the direct, high airflow incubations system of FIG. 3A1 .

This atomizer misting system was trialed with biomass to assess the impact of moisture deposition independent from airflow. Seven parts were loaded into a lab incubator equipped with the atomizer misting system without any airflow (FIG. 5A).

Humidification of this system was achieved by the moisture input into the system via the atomizer.

Two control incubators were run simultaneously using the standard biopolymer humidification system and environmental conditions. One control incubator was set up using the standard direct, high airflow box system and the humidification recirculation system (FIG. 3A1 ) while the other was equipped with only the low, indirect airflow used for the recirculation of humidified air (FIG. 5B). All three incubators were set to standard biopolymer environmental conditions of 99% RH, 5% CO2 and fluctuating temperature of 85-90° F. for nine days of growth.

Direct, high airflow resulted in increased homogeneity of growth within the panels across the entire incubator and allowed the production of the panels of FIG. 1A with minimal differentiation in tissue morphology.

The zero-airflow incubator equipped with the atomizer misting system resulted in highly differentiated panels with a low volume of vertical growth (FIG. 1C). A panel grown by this technique may be characterized in having “bulbs” or bundles of mycelium fibers from 0.1 to 1 inch in diameter and in having discrete dense regions predominantly void of connective tissue.

The low, indirect airflow incubator also resulted in highly differentiated material and reduced aerial growth; however, the volume of vertical growth was increased (FIG. 1B). A panel grown by this technique may be characterized in having “bulbs” or bundles of mycelium fibers equal to or greater than 0.6 inches, for example of from 0.6 to 4 inches in diameter. By comparison, the “bulbs” of mycelium fibers on the panel of FIG. 1C are less than 0.6 inches.

Further, the panel of FIG. 1B is characterized in that the connective tissue is minor and results in a homogeneous aesthetic but heterogeneous performance. This means that, although the surface looks smooth, the mechanical performance may vary through the section of the part.

The high, direct airflow growth environment resulted in panels that were significantly more homogenous, with minimal differentiation throughout the panels (FIG. 1A).

PROCESS STEPS Moisture and Mineral Deposition on Material Surface During Growth

-   1. Nutritious growth media and organism inoculum was packed into     containers 14 as described in US 20150033620 A with the exception     that these containers 14 were not outfitted with lids. -   2. These containers 14 were placed within the incubation chamber 10     maintained under predetermined environmental conditions including     humidity, temperature, carbon dioxide and oxygen. -   3. Moisture and minerals were distributed across the growing surface     of the media in the containers using a bath of water outfitted with     a humidifying puck that atomizes the water into vapor or mist. -   4. Panels were grown for 4 to 14 days within the incubation chamber     10.

Regulation of Moisture and Minerals Within the Substrate to Control Tissue Density

Tests were conducted to determine the effect of regulating the moisture and minerals within a substrate (growth media) prior to incubation in an enclosed incubation chamber with respect to the density of a produced panel of mycological biopolymer.

One test used the following steps:

-   1. Nutritious growth media and organism inoculum was packed into     containers 14 as described in US 20150033620 A with the exception     that these containers 14 are not outfitted with lids. -   2. Moisture and minerals were distributed within the growth media to     achieve a specified moisture between 20-95% moisture. -   3.. Incubating the growth media 15 in each container 14 for a period     of time sufficient to produce a panel of mycelium biopolymer in each     container 14, panels were grown for 4 to 14 days within the     incubation chamber 10.

The result of the test was that the amount of moisture and minerals within the growth media prior to placement in the incubation chamber can be regulated to produce a homogenous panel of mycological biopolymer of a desired density. Of note, moisture contents of 65% on corn stover substrate resulted in densities of 1.7 pcf, and moisture contents of 55% resulted in densities of 2.7 pcf.

In another embodiment, the mycological biopolymer may be grown through a scrim or lofted non-substrate matrix. In this embodiment, the scrim or lofted non-substrate matrix is either organic or inorganic in nature and offers sufficient porosity such that the mycelium can infiltrate the material. The scrim or lofted non-substrate matrix is positioned on or above the nutritive substrate and the entire assembly is incubated in one of the configurations above. The scrim or lofted material serves as reinforcement to the mycelium, a means of oriented and directing tissue growth, a method for consistently removing the grown tissue from the nutritive substrate, or a combination thereof.

In a fourth embodiment, the fluctuation of the percent humidity at time periods of growth throughout the duration of the cycle is employed in order to induce a higher density material of increased homogeneity. In this embodiment, the relative humidity is sustained at a high percentage during the period of aerial mycelium induction, which can begin between day 0 and 5 of growth. Once induced, the humidity is reduced to less than 98% for a period of 4 to 72 hours to induce a densification of the apical tissue. The humidity can then again be elevated to induce newly differentiated growth to provide a range of density, tissue morphology, and orientation through the cross-section of the product. This can be repeated as many times as necessary to garner desired variations in performance through the mycological foam.

In a fifth embodiment, specific air flow rates are used to achieve a range of aerial mycelium densities and mechanical performances. In this embodiment, the air flow can be set at a constant rate, such that the air flow velocity is passively modulated at the tissue grows, or the rate can be adjusted through the course of incubation to deliver a constant rate over the growing tissue. Higher airflow rates have demonstrated the production of denser tissues, while lower airflow rates result in a higher loft of tissue that is less dense when dried. 

1-13. (canceled)
 14. A method of growing a biopolymer material comprising the steps of: providing an incubation chamber; providing growth media within the incubation chamber, wherein the growth media comprises nutritive substrate and a fungus; and growing a mycelium biopolymer consisting essentially of fungal mycelium from the nutritive substrate toa non-substrate reinforcement matrix positioned above or directly contacting the nutritive substrate; and forming a reinforced mycelium biopolymer by further growing the mycelium biopolymer into the non-substrate reinforcement matrix, such that the mycelium biopolymer infiltrates and combines with at least a portion of the non-substrate reinforcement matrix, and the non-substrate reinforcement matrix reinforces the mycelium biopolymer, to form the reinforced mycelium biopolymer.
 15. The method of claim 14, wherein the non-substrate reinforcement matrix comprises at least one of a scrim and a lofted non-substrate matrix.
 16. The method of claim 15, wherein the at least one of the scrim and the lofted non-substrate matrix comprises an organic material.
 17. The method of claim 15, wherein the at least one of the scrim and the lofted non-substrate matrix comprises an inorganic material.
 18. The method of claim 14, further comprising removing the reinforced mycelium biopolymer from the nutritive substrate using the non-substrate matrix.
 19. The method of claim 14, further comprising maintaining the incubation chamber with a predetermined environment of humidity, temperature, carbon dioxide content and oxygen content sufficient to produce the mycelium biopolymer consisting essentially of fungal mycelium.
 20. The method of claim 19, wherein providing growth media within the incubation chamber comprises providing growth media within a container.
 21. The method of claim 20, wherein growing comprises growing the mycelium biopolymer in the container without the use of a lid on the container.
 22. The method of claim 19, further comprising directing flows of air comprising the carbon dioxide content through the incubation chamber and passing the flows of air over the growth media.
 23. The method of claim 22, further comprising incubating the growth media in the incubation chamber for an incubation time period sufficient for the fungus to digest the nutritive substrate and produce the mycelium biopolymer consisting essentially of fungal mycelium.
 24. The method of claim 23, wherein the incubation time period is between 4 and 14 days.
 25. The method of claim 22, wherein the flows of air comprise a carbon dioxide content of 5% to 7% by volume.
 26. The method of claim 22, wherein the flows of air are characterized as having an airflow rate, wherein the airflow rate is between 5 and 10,000 cubic feet per minute.
 27. The method of claim 22, wherein the airflow rate of the flows of air is greater than 160 cubic feet per minute.
 28. The method of claim 27, wherein the airflow rate of the flows of air is greater than 175 cubic feet per minute.
 29. The method of claim 23, wherein the flows of air are pulsed during the incubation time period.
 30. The method of claim 19, further comprising distributing a mist through the incubation chamber for passage over the growth media.
 31. The method of claim 30, wherein the mist comprises moisture and a solute.
 32. The method of claim 31, wherein the solute is selected from a mineral, a carbohydrate, a protein, and a lipid.
 33. The method of claim 32, wherein the solute is a mineral.
 34. The method of claim 33, wherein growing the mycelium biopolymer comprises growing an aerial mycelium, and wherein distributing the mist comprises distributing the mist at regulated amounts onto a top surface of the aerial mycelium.
 35. The method of claim 14, wherein the mycelium biopolymer comprises a density of 0.5 to 4 pounds per cubic foot. 